Summary: Viral spread and seeding of different anatomical compartments within the body depends on cell- to-cell transmission and the ability of infected cells to disseminate by intra- and extravasation. Due to the presence of residual viral replication and occasional reactivation of latent viruses, these processes continue through combination antiretroviral therapy (cART) contributing to viral persistence. In this project, we propose an inducible CRISPR/Cas9 mediated knockout of activated leukocyte cell adhesion molecule (ALCAM/CD166/MEMD) in order to block cell-to-cell HIV-1 transmission and dissemination of infected cells throughout the body. ALCAM is a junctional protein expressed on activated T cells, monocytes and dendritic cells and endothelium. Recent studies show increased levels of ALCAM on CD14+/CD16+ monocytes derived from HIV+ patients and its critical role in promoting transmigration of these cells across the blood brain barrier. Similarly, ALCAM is overexpressed on CD3+ T cells derived from HIV infected patients. Loss of ALCAM disrupted cell aggregation and cell-to-cell HIV transmission. Therefore, ALCAM makes an attractive, non-viral therapeutic target for a gene therapy based approach to treat HIV infected patients. By placing Cas9 endonuclease expression under the control of a minimal Tat dependent HIV-1 derived promoter (5?LTR -80/+66) we created specific, viral expression driven molecular scissors. We designed two sets of paired guide RNAs targeting exons 1 and 14 of the human ALCAM gene. Successful cleavage at the target sites will lead to deletion of the segment of DNA spanning the ALCAM start codon/signal peptide and/or transmembrane domain coding regions, respectively. Our genome editing platform will be active only in cells expressing the viral trans-activator, Tat, i.e., productively infected and reactivated latently infected cells. With successful genome editing, treated cells will lose ALCAM expression and consequently their ability to aggregate and/or transmigrate rendering them unable to spread and transmit the virus to other cells and tissues, including brain. The goal of this novel R21 application is twofold. First, we will characterize the effects of our Tat inducible CRISPR/Cas9-mediated ALCAM knockout on the transmigration activity of the HIV-1 infected monocytes in in vitro settings. Second, we will examine if our specific to infected monocytes/macrophages elimination of ALCAM expression will prevent their dissemination and seeding of various organs/tissue compartments in humanized mice model.